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Methods for Setting Up Primary Cultures Specific to Animal Groups
Subprotocol 2
Sea Urchin (Echinoidea) Embryos
Materials
Equipment - Tissue culture dish (100 x 20 mm; Falcon, Becton-Dickinson)
- Hand centrifuge
- Centrifuge and centrifuge tubes
- Nitex filter (20 and 64 !-1m) (Tetko Inc.)
Solutions - ASW (Instant Ocean; Aquarium Systems)
- FSW (seawater filtered through a 0.45-!-Im pore membrane filter)
- Culture medium (FSW 20 ml, horse serum 4%, gentamicin 50 !-Ig/ml)
Miscellaneous - Trypan blue (Sigma)
- Horse serum (Gibco)
Animals - Sea urchin Strongylocentrotus purpuratus
Procedure
Fertilization 1. Remove matured eggs from sea urchin ovaries (Fig. 3).
2. Wash the sea urchin eggs three times with FSW using a hand centrifuge.
3. Resuspend the pellet in FSW.
4. Fertilize the eggs by the addition of concentrated sperm in FSW.
5. Wash the zygotes three times until free of sperm.
6. Maintain the zygotes in FSW.
7. Harvest the embryos at the mesenchyme blastula stage (20-32 h after
fertilization) by centrifugation in a hand centrifuge.
Dissociation 1. Wash the pelleted embryos by resuspension in 160 ml of Ca2+ - and
Mg2+ -free seawater.
2. Pass the suspension through a 64-!-Im Nitex filter.
3. After 5 min at 4°C, dissociate the remaining cell aggregates in the eluate by passing them through a 20-!-Im Nitex filter.
4. Centrifuge the last eluate at 1,000 g for 2 min at 4°C.
Culture 1. Resuspend the pellet in FSW to a final volume of 10-12 ml in a centriset-up
fuge tube by gentle swirling of the tube.
2. Check the cell viability with Trypan blue.
3. Plate the cell suspension in the culture medium in culture dishes previously washed with culture medium.
Methods for Setting Up Primary Cultures Specific to Animal Groups
Subprotocol 2
Sea Urchin (Echinoidea) Embryos
Materials
Equipment - Tissue culture dish (100 x 20 mm; Falcon, Becton-Dickinson)
- Hand centrifuge
- Centrifuge and centrifuge tubes
- Nitex filter (20 and 64 !-1m) (Tetko Inc.)
Solutions - ASW (Instant Ocean; Aquarium Systems)
- FSW (seawater filtered through a 0.45-!-Im pore membrane filter)
- Culture medium (FSW 20 ml, horse serum 4%, gentamicin 50 !-Ig/ml)
Miscellaneous - Trypan blue (Sigma)
- Horse serum (Gibco)
Animals - Sea urchin Strongylocentrotus purpuratus
Procedure
Fertilization 1. Remove matured eggs from sea urchin ovaries (Fig. 3).
2. Wash the sea urchin eggs three times with FSW using a hand centrifuge.
3. Resuspend the pellet in FSW.
4. Fertilize the eggs by the addition of concentrated sperm in FSW.
5. Wash the zygotes three times until free of sperm.
6. Maintain the zygotes in FSW.
7. Harvest the embryos at the mesenchyme blastula stage (20-32 h after
fertilization) by centrifugation in a hand centrifuge.
Dissociation 1. Wash the pelleted embryos by resuspension in 160 ml of Ca2+ - and
Mg2+ -free seawater.
2. Pass the suspension through a 64-!-Im Nitex filter.
3. After 5 min at 4°C, dissociate the remaining cell aggregates in the eluate by passing them through a 20-!-Im Nitex filter.
4. Centrifuge the last eluate at 1,000 g for 2 min at 4°C.
Culture 1. Resuspend the pellet in FSW to a final volume of 10-12 ml in a centriset-up
fuge tube by gentle swirling of the tube.
2. Check the cell viability with Trypan blue.
3. Plate the cell suspension in the culture medium in culture dishes previously washed with culture medium.